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1.
Production and application of a polyclonal peptide antiserum for universal detection of StAR protein 总被引:1,自引:0,他引:1
We report production of a polyclonal antibody against the StAR (steroidogenic acute regulatory) protein of steroidogenic cells and immunohistochemistry (IHC) staining of bovine adrenal gland tissue available in paraffin block. The epitope-specific polyclonal antibody was produced in a rabbit immunized against a synthetic 26 amino acid peptide (82AMQRALGILKDQEGWKKESRANGDE107) derived from the coding sequences reported for the bovine StAR gene (Gene Bank Accession No. Q28918). Western blots were developed using the StAR-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The antiserum was found to be highly specific for StAR, which exhibited an estimated molecular weight of about 30 KDa for all species analyzed. Finally, the peptide antiserum was successfully employed to localize StAR protein by immunohistochemical staining of thin sections prepared from bovine adrenal gland tissue. This study is the first to report a polyclonal peptide antiserum that apparently recognizes native StAR protein, regardless of the species of origin. The successful production of the antibody has provided a useful tool for studying regulation of StAR protein. 相似文献
2.
Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody purification, the antibodies are characterized based on their affinity or specificity. An efficient approach for characterization of peptide antibodies is epitope mapping using peptide based assays. This review describes standard solid-phase approaches for generation of peptide antibodies with special emphasis on peptide selection, generation of peptide conjugates for immunization and characterization of the resulting peptide antibodies. 相似文献
3.
Douglas C. Marchion Donald S. Manning William M. Shafer Ralph C. Judd 《Molecular biotechnology》1996,6(3):231-240
The ability to prevent disease by immunization with subunit vaccines that incorporate specific epitopes was demonstrated by
DiMarchi et al. (1), who used a synthetic peptide to protect cattle against foot-and-mouth disease. However, generation of antibody to peptide
antigens is often difficult owing to the small molecular mass and limited chemical complexity. We tested the hypothesis that
recombinant DNA and synthetic peptide techniques would make it possible to stimulate vigorous immune responses to specific
epitopes of an outer membrane protein ofNeisseria gonorrhoeae.
The MtrC AP1 sequence from the invariant mtrC gonococcal lipoprotein was genetically fused to maltose binding protein. The
resultant fusion protein was used as the primary immunogen to stimulate MtrC AP1-specific antiserum. To enhance antibody production
specific to MtrC AP1, boosting immunizations were performed with synthetic MtrC AP1 sequence contained in a multiple antigenic
peptide system immunogen. The MtrC AP1-specific antiserum strongly recognized the MtrC protein on Western blots and appeared
to bind native MtrC proteinin situ. The generation of antibody in this fashion provides the technology to produce antibody to defined epitopes of any protein,
including those found in the gonococcal outer membrane. The ability of those antibodies to inhibit bacterial growth or to
activate complement protein can then be tested. 相似文献
4.
A Galvan P D Lampe K C Hur J B Howard E D Eccleston M Arneson C F Louis 《The Journal of biological chemistry》1989,264(33):19974-19978
The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase. 相似文献
5.
重组抗体药物在抗肿瘤、抗自身免疫病、抗感染等领域都有较好的应用前景,且市场的需求量正在逐年上升。然而许多的抗体药物在研究、生产过程中都面临着产量低、稳定性差、活性低等问题。目前,在大规模制备抗体药物的过程中,常常利用动物细胞表达系统来生产分泌蛋白,而信号肽作为连接在分泌蛋白N端的一段氨基酸序列,能够控制蛋白质的分泌途径进而影响抗体蛋白的表达产量。简要概述了信号肽的一级结构与作用机制,同时,指出其对重组抗体蛋白分泌效率的影响途径,最后结合各种研究成果总结出信号肽序列优化策略。 相似文献
6.
Kameshita I Kinoshita S Shigeri Y Tatsu Y Yumoto N Ishida A 《Journal of biochemical and biophysical methods》2004,60(1):13-22
In order to obtain a polyclonal antibody that recognizes various protein kinases, a peptide corresponding to an amino acid sequence of a highly conserved subdomain (subdomain VIB) of the protein kinase family was synthesized and used for immunization. When the synthetic peptide, CVVHRDLKPENLLLAS, was coupled to keyhole limpet hemocyanin (KLH) and used to immunize rabbits, polyclonal antibodies that detected multiple protein kinases on a Western blot were generated. One of the antibodies obtained, KI98, detected a variety of purified Ser/Thr protein kinases, such as calmodulin-dependent protein kinase II (CaM-kinase II), calmodulin-dependent protein kinase IV (CaM-kinase IV), cAMP-dependent protein kinase, protein kinase C, and Erk2. The antibody detected as low as 0.2 ng of protein kinases blotted onto a nitrocellulose membrane by dot-immunobinding assay. When a rat brain extract was analyzed with this antibody, various protein kinases were simultaneously detected. The present anti-peptide antibody with a broad spectrum of cross-reactivity to multiple protein kinases may be a powerful tool for comprehensive analysis focused on protein kinases. 相似文献
7.
Anti-peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex 总被引:1,自引:1,他引:0
Gerard Peaucellier Kirsten Shartzer Wanping Jiang Katia Maggio William H. Kinsey 《Development, growth & differentiation》1993,35(2):199-208
The egg plasma membrane and cortical structures are highly enriched in protein tyrosine kinase activity which is thought to play an important role in the fertilization process. In order to identify the tyrosine protein kinases in the egg cortex, a site directed polyclonal antibody was produced against a peptide duplicating a conserved region of the catalytic domain of the sea urchin c-abl gene product. The region chosen as an antigen had a high degree of homology (57%) to other protein tyrosine kinases. The antibody was found to bind with a high degree of specificity to a 57 kDa protein tyrosine kinase in S. purpuratus eggs. The antibody was capable of immunoprecipitating the enzyme as a 57 kDa phosphoprotein from purified egg cortex fractions solubilized in NP-40. Immunoprecipitation was completely inhibited by prior incubation of the antibody with the synthetic peptide used as an antigen. Binding of the antibody completely inhibited kinase activity. However, the immunoprecipitated kinase activity could be eluted from the Sepharose-coupled antibody and was shown to have catalytic activity towards a tyrosine containing peptide substrate. The enzyme also underwent autophosphorylation on tyrosine in vitro. Ultrastructural localization of the kinase by immuno-electron microscopy revealed that the enzyme was primarily restricted to the egg plasma membrane. 相似文献
8.
Choulier L Rauffer-Bruyère N Ben Khalifa M Martin F Vernet T Altschuh D 《Biochemistry》1999,38(12):3530-3537
Monoclonal antibody 57P, which was raised against tobacco mosaic virus protein, cross-reacts with a peptide corresponding to residues 134-146 of this protein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics the structure in the protein. In this study, we carried out a detailed comparison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the parent protein, and the effect of these substitutions on antibody binding parameters have been measured with a Biacore instrument. Fabs that recognize epitopes located away from the site of mutations were used as indirect probes for the conformational integrity of protein antigens. Their interaction kinetics with all proteins were similar, suggesting that the substitutions had no drastic effect on their conformation. The five substitutions introduced in the peptide and the protein had minor effects on association rate constants (ka) and significant effects on dissociation rate constants (kd) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope was presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation. However, ka values were about 10 times larger and kd values about 5 times larger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed for three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and protein antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to mimic correctly some but not all properties of the protein-Fab 57P interaction and highlight the importance of quantitative analysis of both equilibrium and kinetic binding parameters in the design of synthetic vaccines and drugs. 相似文献
9.
K Hilpert G Hansen H Wessner G Küttner K Welfle M Seifert W H?hne 《Protein engineering》2001,14(10):803-806
The 9E10 antibody epitope (EQKLISEEDL) derives from a protein sequence in the human proto-oncogen p62(c-myc) and is widely used as a protein fusion tag. This myc-tag is a powerful tool in protein localization, immunochemistry, ELISA or protein purification. Here, we characterize the myc-tag epitope by substitutional analysis and length variation using peptide spot synthesis on cellulose. The key amino acids of this interaction are the core residues LISE. The shortest peptide with a strong binding signal is KLISEEDL. Dissociation constants of selected peptide variants to the antibody 9E10 were determined. scFv constructs with the shortest possible myc-tags were successfully detected by Western blot and ELISA, giving a signal comparable to that of the original myc-tag. 相似文献
10.
A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response. 总被引:2,自引:1,他引:1
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A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses. 相似文献
11.
In our effort to develop synthetic immunogens as vaccines, we have focused on the combination of a known T-cell stimulating peptide with putative B-cell stimulating peptide epitopes derived from the sequences of respiratory syncytial (RS) virus proteins. The T-cell stimulating peptide consists of residues 45 through 60 of the 1A protein of RS virus, and it also contains an overlapping antibody binding (B-cell) site. Herein, we have combined the 1A T-cell stimulating peptide with a putative B-cell peptide epitope derived from the viral G glycoprotein using linear synthesis or using chemical crosslinking. The chimeric immunogens were compared to each other and to free peptides for their T- and B-cell stimulating properties. Both chimeras had potent T-cell stimulating and antibody-inducing activity. However, T-cells primed to free peptide differentially recognized the two chimeras and immunization with the chimeras primed T-cells with different specificity. Most strikingly, the two chimeras had opposite antibody-inducing properties: The chimera constructed by linear synthesis overwhelmingly elicited antibody directed against the G peptide, whereas the chimera constructed by chemical crosslinking overwhelmingly elicited antibody directed against the 1A peptide. Competition blocking studies revealed that the chimeras adopted different configurations in solution. The resulting antibody response, and hence the B-cell clone elicited, was consistent with the antibody accessibility of the individual peptide epitope. 相似文献
12.
Allosteric activation of 5(')-AMP-activated protein kinase (AMPK) is currently of interest as an approach for the treatment of metabolic disorders because AMPK plays multiple roles in glucose and lipid metabolism. The availability of ultrafast, ultrasensitive, and robust assays suited to high-throughput screening (HTS) is key to obtaining small-molecule AMPK activators. In the absence of high-affinity and selective antiphospho Ser/Thr antibodies for AMPK substrates, we have developed two homogeneous AMPK assays with the commercially available antibody Anti-pS(133)-CREB and an engineered peptide ACC-CREBp. Anti-pS(133)-CREB antibody was raised against the phospho-CREB peptide derived from cAMP response element binding protein (CREB). ACC-CREBp was a variant (Arg to Pro) of ACC-CREB, a hybrid peptide consisting of a 9-amino-acid peptide from rat acetyl-CoA carboxylase (ACC), CREB peptide, and the addition of two hydrophobic Leu residues. ACC-CREBp showed increased suitability as a substrate for AMPK, eliminated phosphorylation by PKA, and preserved antibody binding. The homogeneous time-resolved fluorescence and AlphaScreen AMPK assays were developed using both Anti-pS(133)-CREB antibody and ACC-CREBp that are either labeled with a fluorescent probe or linked to a photoactivated bead, respectively. Thus, ACC-CREBp phosphorylation can be measured as a signal change resulting from the formation of antibody-antigen complex. This approach of adapting known antibody and antigenic peptide pairs to monitor alternate Ser/Thr kinases may be of general use. 相似文献
13.
Interaction of monoclonal anti-peptide antibodies with lysozyme 总被引:2,自引:0,他引:2
The interaction of monoclonal anti-peptide antibodies with the free peptide and its protein counterpart has been evaluated for hen egg white lysozyme and the peptide constituting residues 38 to 45. Fluorescence methodology has been developed for the measurement of association constants based on resonance energy transfer between the excited tryptophan of antibody and bound peptide ligand conjugated to a fluorescent probe. Five antibodies, four IgM and one IgG, have been assayed by ELISA, and have demonstrated binding to the adsorbed peptide alone, to the adsorbed lysozyme alone, or to both. Multivalent interaction with the adsorbed ligand is a key factor in the efficacy of binding. Measurement of binding constants in homogeneous solution, by equilibrium dialysis and energy transfer, demonstrated that lysozyme was bound to an IgG antipeptide antibody with an association constant (4 X 10(2) M-1) 200-fold less than that for the free peptide (8 X 10(4) M-1). It was also inferred for IgM that an association constant of the order of 10(2) M-1 was sufficient to effect selective interaction in a system providing multivalent interaction. The shared conformations between protein and peptide, implied by the specific reactivity of the anti-peptide antibody with the protein, points to structural fluctuations of the surface regions and residues of globular proteins. 相似文献
14.
Epitopes of peptide 43-88 of guinea pig myelin basic protein: localization with monoclonal antibodies 总被引:8,自引:0,他引:8
Two monoclonal antibodies, one raised by immunization with mouse myelin basic protein (MBP) and the second raised by immunization with peptide 68-88 of guinea pig MBP, were compared with respect to specificity. The former antibody (15.32) cross-reacted completely with rat, guinea pig, human, and bovine MBP. It also reacted with peptide 43-88 from each MBP. The latter antibody (22.17) was nonreactive with MBP, but cross-reacted with peptide 43-88 from rat, human, guinea pig, and bovine MBP. When tested with small peptides derived from peptide 43-88, antibody 22.17 reacted with an epitope in the C-terminal region. Antibody 15.32 reacted with an epitope in the N-terminal half of the peptide. The data show that 22.17 reacted with a unique epitope associated only with free peptide, whereas 15.32 recognized an epitope common to both peptide 43-88 and MBP. 相似文献
15.
Karasavvas N Beck Z Tong J Matyas GR Rao M McCutchan FE Michael NL Alving CR 《Biochemical and biophysical research communications》2008,366(4):982-987
Natural polyreactive antibodies can accommodate chemically unrelated epitopes, such as lipids and proteins, in a single antigen binding site. Because liposomes containing lipid A as an adjuvant can induce antibodies directed against specific lipids, we immunized mice with liposomes containing lipid A together with a protein or peptide antigen to determine whether monoclonal antibodies generated after immunization would be specifically directed both to the liposomal lipid (either cholesterol or galactosylceramide) and also to the accompanying liposomal protein or peptide. Monoclonal antibodies were obtained that bound, by ELISA, to cholesterol and to recombinant gp140 envelope protein from HIV-1, or to galactosylceramide and to an HIV-1 envelope peptide. Surface plasmon resonance studies with the former antibody showed that the liposomal cholesterol and liposomal gp140 each contributed to the overall binding energy of the antibody to liposomes containing cholesterol and protein. 相似文献
16.
A Malley L Werner E Benjamini C Y Leung J Torres N Pangares S Shiigi M Axthelm 《Journal of medical primatology》1991,20(4):177-181
Synthetic envelope peptides of a simian retrovirus (SRV-2) were used to define both T- and B-cell epitopes of the envelope protein. The SRV-2 peptide 100-106 specifically blocks rhesus anti-SRV-2 neutralizing antibody activity, and a peptide 100-106 keyhole limpet hemocyanin conjugate induces a strong antipeptide antibody response. SRV-2 peptide 100-106 and 233-249 induces good T-cell proliferation of murine spleen cells immunized with the SRV-2 virus. Thus, SRV-2 envelope peptide 100-106 represents both a T- and B-cell epitope, and peptide 233-249 a T-cell epitope. 相似文献
17.
Three kinds of biotinylated peptides with different linkers between biotin and beta-sheet peptide were designed and synthesized. The transmission electron microscopy revealed that the biotinylated peptides self-assembled to form a tubular structure with external diameter of ca. 60 nm and inner diameter of ca. 30 nm in an aqueous solution. The anti-biotin antibody effectively bound to biotin groups in the peptide nanotubes. The binding of antibody was regulated by not only the concentration of the protein in the solution but also the properties of biotinylated peptides forming the tubes. The antibody preferentially bound to the biotinylated peptide tubes assembled from the peptide with the most hydrophilic linker, suggesting that the surface properties and functions of the tubular structure were modulated and engineered by the design of the peptides. 相似文献
18.
S Higashi S Kawabata H Nishimura H Funasaki S Ohyama S Miyamoto A Funatsu S Iwanaga 《Journal of biochemistry》1990,108(4):654-662
A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor VIIa in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Kd = 1.12 x 10(-10)M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2(+)-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a gamma-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII. 相似文献
19.
Priming for rotavirus neutralizing antibodies by a VP4 protein-derived synthetic peptide. 总被引:7,自引:2,他引:5
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In the rotavirus SA11 surface protein VP4, the trypsin cleavage sites associated with the enhancement of infectivity are flanked by two amino acid regions that are highly conserved among different rotaviruses. We have tested the ability of synthetic peptides that mimic these two regions to induce and prime for a rotavirus neutralizing antibody response in mice. After the peptide immunization schedule, both peptides induced peptide antibodies, but neither was able to induce virus antibodies, as measured by an enzyme-linked immunosorbent assay or a neutralization assay. However, when the peptide-inoculated mice were subsequently injected with intact SA11 virus, a rapid and high neutralizing antibody response was observed in mice that had previously received the peptide comprising amino acids 220 to 233 of the VP4 protein. This neutralizing activity was serotype specific; however, this peptide was also able to efficiently prime the immune system of mice for a neutralizing antibody response to the heterotypic rotavirus ST3 when the ST3 virus was used for the secondary inoculation. 相似文献
20.
S M Farooqui C Prasad M Ali 《Biochemical and biophysical research communications》1992,184(2):661-667
A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein. 相似文献